The general objective of the proposed research in this renewal application is to investigate the role of the inducible isoform of nitric oxide synthase (iNOS) in the biochemistry and pathobiology of cardiac allograft rejection. The central hypothesis to be tested is that NO produced by iNOS in macrophages infiltrating the myocardium and in the cardiac myocytes augments the myocardial inflammation and contributes to the death of cardiac myocytes. We have demonstrated: 1) that iNOS mRNA, protein, and enzyme activity are induced in endothelial cells, infiltrating macrophages and cardiomyocytes in rejecting cardiac allografts, and 2) that iNOS induction is accompanied by impaired ventricular function and death of heart muscle cells which occurs both by necrosis and by apoptosis. We now propose to investigate mechanisms responsible for necrosis, apoptosis, and iNOS expression during heart transplant rejection. Aim #1 is to investigate, using cultured cardiomyocytes and rat and mouse heterotopic cardiac transplantation models, the hypothesis that activation of polyadenosine 5' -diphosphoribose synthetase (PARS) by nitric oxide contributes to the necrosis of cardiac myocytes in vitro and during cardiac allograft rejection. Aim #2 is to investigate the hypothesis that myocardial inflammation, necrosis and apoptosis during cardiac allograft rejection are ameliorated using mice as allograft donors and recipients that are unable to express iNOS (iNOS-ko mice). Aim#3 is to investigate the hypotheses that apoptosis of cardiomyocytes triggered by NO can be inhibited by sem-selective iNOS inhibitors, by transfection with Bcl-2, and by administration of caspase inhibitors. Aim #4 is to investigate the interplay between iNOS and COX-2 in modulating prostaglandin and thromboxane synthesis during cardiac allograft rejection, the role of CD154-CD40 interaction in the expression of iNOS and COX-2 in cardiomyocytes and the effect of COX-2 expression on cardiomyocyte apoptosis in cardiac allograft rejection. The proposed experiments may provide new insights concerning the role of iNOS in pathobiology and potential therapy of cardiac allograft rejection and they may also be relevant to other cardiac diseases in which iNOS is expressed such as myocardial infarction and dilated cardiomyopathy.